Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) are encoded by the carboxyterminal portion of viral open reading frame 3

Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were revealed by serologic analysis of a recombinant protein derived from PRRSV open reading frame 3 (ORF 3). The hydrophilic carboxyterminal 199 amino acids encoded by the ORF 3 of a European (Lelystad) isolate of PRRSV were expressed as a recombinant fusion protein (BP03-P) in a baculovirus gene expression system. Sera from gnotobiotic swine exposed to prototypic reference European and American isolates of PRRSV and sera from conventionally reared European and American swine convalescing from naturally acquired PRRSV infections were used to characterize the BP03-P protein. Sera from gnotobiotic and conventionally reared swine exposed to European isolates of PRRSV were significantly more reactive (P < 0.01) with BP03-P than were the corresponding American PRRSV antisera using the indirect immunoperoxidase monolayer assay (IPMA). Prototypic European, but not American, PRRSV antisera also recognized BP03-P using western immunoblotting and radioimmunoprecipitation assay (RIPA) procedures. However, gnotobiotically derived antiserum to an atypical American-origin PRRSV was reactive with BP03-P by both IPMA and western immunoblot. Despite a predicted potential for N-linked glycosylation, studies with tunicamycin and peptide-N-glycosidase F (PNGase F) indicated that BP03-P was not N-glycosylated in either insect cell cultures or Trichoplusia ni larvae infected with the recombinant baculovirus. Sera from rabbits inoculated with BP03-P failed to neutralize both the European (Lelystad) and American (ATCC VR-2332) reference isolates of PRRSV and did not react by IPMA with PRRSV-infected cell cultures. Taken together, the data suggest that the carboxyterminal portion of PRRSV ORF 3 encodes a non-neutralizing viral peptide that is partially responsible for the serologic differences noted between European and most American isolates of PRRSV.     

Monoclonal antibodies against conformationally dependent epitopes on porcine reproductive and respiratory syndrome virus

Monoclonal antibodies (MAbs) against porcine reproductive and respiratory syndrome virus (PRRSV) were prepared and characterized. Four MAbs were developed from the mice immunized with the recombinant GP4 protein expressed in insect cells, and six MAbs were derived from the immunization with recombinant GP5 protein. All of the MAbs showed strong perinuclear fluorescence in PRRSV VR2385 infected cells by immunofluorescence staining. Among the MAbs to GP5 protein, one showed strong reactivity in ELISA and recognized a 26 kDa band of PRRSV in a western blot assay, while another showed neutralizing activity against the VR2385 isolate. Out of the four MAbs to GP4 protein, one showed mild reactivity in ELISA with detergent extracted antigen, but had no reactivity in a western-blot assay. The failure of MAb binding to detergent extracted antigen in ELISA or in western-blot analysis indicated that the MAbs were against conformationally dependent epitopes. Reactivity patterns of the MAbs with PRRSV field isolates tested by fixed-cell ELISA showed that there are antigenic variations in PRRSV GP4 and GP5 proteins. Development of these MAbs will benefit further studies on PRRSV structural proteins as well as in understanding their roles in PRRSV pathogenesis.     

Expression cloning and humoral immune response to the nucleocapsid and membrane proteins of equine arteritis virus

To provide a convenient and sensitive method for the detection of equine arteritis virus (EAV)-specific serum antibodies, we developed an immunoblot assay employing the EAV nucleocapsid (N) and membrane (M) proteins expressed in a procaryotic expression vector (pMAL-c2) for the production of recombinant maltose-binding (MBP) fusion proteins (MBP-N and MBP-M). The antigenic reactivity of the recombinant fusion proteins and their Xa factor cleavage EAV products was confirmed by immunoblot using horse antisera to EAV. Some horse sera, however, showed immune reactivity to the MBP fusion partner protein. Based on a total of 32 horse sera analyzed for the presence of EAV antibodies by immunoblot, using the MBP-N or -M fusion proteins and the Xa factor cleavage EAV products, and in the serum neutralization test, there was 100% concordance between the assays. Sera from horses experimentally infected with EAV were reactive in the immunoblot test with both the MBP-N and the MBP-M fusion proteins by day 14 after EAV exposure. The reactivity continued to the end of the experiment at day 145 after infection. This immune reactivity correlated with the detection of neutralizing antibodies in the serum samples. Based on these findings, the recombinant N and M proteins might be useful for serodetection of EAV-infected animals.     

Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus

GP4 is a minor structural glycoprotein encoded by ORF4 of Lelystad virus (LV). When it was immunoprecipitated from cell lysates and extracellular virus of CL2621 cells infected with LV, it was shown to have an apparent molecular mass of approximately 28 and 31 kDa, respectively. This difference in size occurred because its core N-glycans were modified to complex type N-glycans during the transport of the protein through the endoplasmic reticulum and Golgi compartment. A panel of 15 neutralizing monoclonal antibodies (MAbs) reacted with the native GP4 protein expressed by LV and the recombinant GP4 protein expressed in a Semliki Forest virus expression system. However, these MAbs did not react with the GP4 protein of U.S. isolate VR2332. To map the binding site of the MAbs, chimeric constructs composed of ORF4 of LV and VR2332 were generated. The reactivity of these constructs indicated that all the MAbs were directed against a region spanning amino acids 40 to 79 of the GP4 protein of LV. Six MAbs reacted with solid-phase synthetic dodecapeptides. The core of this site consists of amino acids 59 to 67 (SAAQEKISF). Comparison of the amino acid sequences of GP4 proteins from various European and North American isolates indicated that the neutralization domain spanning amino acids 40 to 79 is the most variable region of GP4. The neutralization domain of GP4, described here, is the first identified for LV.     

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.     

Synthesis and function of Actinomyces naeslundii T14V type 1 fimbriae require the expression of additional fimbria-associated genes

The nucleotide sequence of the chromosomal DNA flanking the Actinomyces naeslundii (formerly A. viscosus) T14V type 1 fimbrial structural subunit gene (fimP) was determined. Six open reading frames (ORFs), in the order 5' ORF3, ORF2, ORF1,fimP, ORF4, ORF5, ORF6 3', were identified. ORF1 encoded a protein of 408 amino acid residues (Mr = 39,270) and had significant sequence homology with the A. naeslundii T14V type 1 and A. naeslundii WVU45 type 2 fimbrial structural subunits. An in-frame fusion of ORF1 to the malE gene of the expression vector, pMAL-c2, yielded a protein that was immunostained with antibodies raised against the maltose binding protein and A. naeslundii T14V whole bacteria. Digestion of the fusion protein with factor Xa released a protein (apparent molecular mass of 34 kDa) that was immunostained only with the antibody directed against A. naeslundii T14V whole bacterial cells. Integration plasmids carrying a kanamycin resistance gene (kan) that was used to substitute for ORF1 or for DNA fragments internal to the coding region of the other five ORFs were used to transform A. naeslundii T14V. Neither type 1 fimbriae nor the 65-kDa fimbrial structural subunit was detected in mutants obtained by allelic replacement of ORF1 or ORF2. Mutants obtained by allelic replacement of ORF3 or ORF4 expressed only the 65-kDa fimbrial structural subunit. These mutants did not bind, in vitro, to proline-rich proteins that serve as the receptors for Actinomyces type 1 fimbriae. In contrast, a mutant in which the integration plasmid DNA had been inserted at a site close to the carboxyl terminus of ORF6 expressed type 1 fimbriae and had adherence properties similar to those observed in the wild-type strain. These results demonstrate the existence of additional genes near fimP that are likely to be involved in the synthesis and function of cell surface fimbriae of A. naeslundii T14V.     

The Epstein-Barr virus (EBV) gN homolog BLRF1 encodes a 15-kilodalton glycoprotein that cannot be authentically processed unless it is coexpressed with the EBV gM homolog BBRF3

The Epstein-Barr virus (EBV) homolog of the conserved herpesvirus glycoprotein gN is predicted to be encoded by the BLRF1 open reading frame (ORF). Antipeptide antibody to a sequence corresponding to residues in the predicted BLRF1 ORF immunoprecipitated a doublet of approximately 8 kDa from cells expressing the BLRF1 ORF as a recombinant protein. In addition, four glycosylated proteins of 113, 84, 48, and 15 kDa could be immunoprecipitated from virus-producing cells by the same antibody. The 15-kDa species was the mature form of gN, which carried alpha2,6-sialic acid residues. The remaining glycoproteins which associated with gN were products of the BBRF3 ORF of EBV, which encodes the EBV gM homolog. The 8-kDa doublet seen in cells expressing recombinant gN comprised precursors of the mature 15-kDa gN. Coexpression of EBV gM with EBV gN was required for authentic processing of the 8-kDa forms to the 15-kDa form.     

A Moloney murine leukemia virus-based retroviral vector pseudotyped by the insect retroviral gypsy envelope can infect Drosophila cells

The gypsy element of Drosophila melanogaster is the first retrovirus identified so far in invertebrates. Previous data suggest that gypsy ENV-like ORF3 mediates viral infectivity. We have produced in the 293GP/LNhsp701ucL.3 human cell line a Moloney murine leukemia virus-based retroviral vector pseudotyped by the gypsy ENV-like protein. We have shown by immunostaining that the gypsy envelope protein is produced in 293GP/LNhsp701ucL.3 cells and that vector particles collected from these cells can infect Drosophila cells. Our results provide direct evidence that the infectious property of gypsy is due to its ORF3 gene product.     

Addition of cisapride shortens colonoscopy preparation with lavage in elderly patients

The major envelope protein, p35, of vaccinia virus (VV; strain LIVP) was purified by extraction from virions with the non-ionic detergent Nonidet P-40. The protein was cleaved with CNBr. Four homogeneous peptides were isolated and their N-terminal amino-acid (aa) sequences determined. A computer search of a protein sequence databank revealed complete identity of the determined sequences with aa 44-63, 144-149, 154-165 and 224-238 of ORF H3 of the HindIII-H fragment of the VV genome [Rosel et al., J. Virol. 60 (1989) 436-446]. Earlier, Gordon et al. [Virology 167 (1988) 361-369] determined that the p35 surface protein of VV strain IHD-W is encoded by the H6 gene. Muravlev et al. [Biopolymery i kletka 6 (1990) 83-89 (Russian)] deduced from their data that gene A2 encodes this prominent antigen. Taking into account this ambiguity, we cloned the genes H3, H6 and A2 in expression vectors, prepared the specific antisera against the expression products and conducted the immunochemical analysis of the recombinant and native VV-specific proteins. It has been established that the H6 codes for an early protein that is found only in the infected cell extracts, but is absent in mature virions. The immunodominant protein p35 of VV strain LIVP is encoded by the gene H3. The gene A2 protein product is present mainly in the infected cell extract, but the antiserum against the A2 product shows a rather weak interaction with the 35-kDa fraction of structural VV proteins resolved by electrophoresis.     

Banana bunchy top virus DNA-3 encodes the viral coat protein

Banana bunchy top virus (BBTV) has a multicomponent genome consisting of at least six circular single-stranded DNAs each with a single large open reading frame (ORF) in the virion sense. A protein of approximately 20 kDa has been associated with purified virions and is assumed to be the viral coat protein. The N-terminus of this protein was sequenced and compared to the predicted amino acid sequence of the large ORF of BBTV DNA-1 to 6. This comparison indicated that the ORF of BBTV DNA-3, which potentially encodes a protein of 19.3 kDa, was the coat protein gene of BBTV. The ORF sequence of BBTV DNA-3 was cloned into a prokaryotic expression vector, pMAL-c2, and the resulting maltose binding-BBTV coat protein fusion product was purified and used for the production of polyclonal antiserum in a rabbit. The resultant antiserum was able to detect the presence of BBTV in infected leaf tissue confirming that the large virion sense ORF of BBTV DNA-3 encodes the viral coat protein.     

The adenylyl and guanylyl cyclase superfamily

Antibodies and their recombinant fragments have enormous potential for therapy of malignant and other diseases, but there can be problems associated with their production and purification in the quantities required for therapeutic use. We investigated the use of gene therapy for the production of such recombinant antibody fragments in vivo. We generated two recombinant adenoviruses expressing the single chain Fvs (scFvs) fused to murine GM-CSF (mGM-CSF). The scFvs used are MFE-23 which binds to a human tumour-associated marker carcino-embryonic antigen (CEA) and B1.8 which binds the hapten 4-hydroxy-3-nitro-5-iodo-phenylacetyl (NIP). Using scFvs to target GM-CSF to tumour cells should reduce the systemic toxicity of GM-CSF but retain its ability as a cytokine to induce systemic immune responses to tumour targets. Cell lines infected with the recombinant adenoviruses in vitro express and secrete high levels of the scFv.mGM-CSF fusion proteins. The scFv retains specificity while the mGM-CSF portion is fully bioactive and there is no detectable degradation of the fusion product. We also demonstrated effective in vivo expression of the scFv.mGM-CSF proteins. C57BI/6 mice injected intravenously with the adenovirus encoding the MFE-23.mGM-CSF fusion produce high levels of the fusion protein by 2 days after infection. The scFv.mGM-CSF protein can be detected in the serum, at biologically active levels, for at least 20 days and the level of protein produced is related to the amount of adenovirus injected. This approach has the potential to streamline the testing of the many therapeutic strategies based on recombinant scFvs and we are currently testing these constructs in an animal model for antitumour activity.     

Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.     

Production of recombinant human brain type I inositol-1,4,5-trisphosphate 5-phosphatase in Escherichia coli. Lack of phosphorylation by protein kinase C

The dephosphorylation of inositol 1,4,5-trisphosphate (InsP3) to inositol 1,4-bisphosphate is catalyzed by InsP3 5-phosphatase. The coding region of human brain type I InsP3 5-phosphatase was expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The relative molecular mass of the purified fusion protein (MBP-InsP3-5-phosphatase) was approximately M(r) 85,000 as analysed by SDS/PAGE. The yield was about 10 mg fusion protein/l lysate. After cleavage from MBP with factor Xa, the specific activity of recombinant 5-phosphatase was 120-250 mumol.mg-1.min-1. The molecular mass of purified protein by SDS/PAGE was M(r) 43,000. The activity was inactivated by p-hydroxymercuribenzoate. The possibility that protein kinase C might phosphorylate InsP3 5-phosphatase was tested on the purified 43,000 M(r) protein. In this study, we show that recombinant 5-phosphatase is not a substrate of protein kinase C.     

Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently described arterivirus responsible for disease in swine worldwide. Comparative sequence analysis of 3'-terminal structural genes of the single-stranded RNA viral genome revealed the presence of two genotypic classes of PRRSV, represented by the prototype North American and European strains, VR-2332 and Lelystad virus (LV), respectively. To better understand the evolution and pathogenicity of PRRSV, we obtained the 12,066-base 5'-terminal nucleotide sequence of VR-2332, encoding the viral replication activities, and compared it to those of LV and other arteriviruses. VR-2332 and LV differ markedly in the 5' leader and sections of the open reading frame (ORF) 1a region. The ORF 1b sequence was nearly colinear but varied in similarity of proteins encoded in identified regions. Furthermore, molecular and biochemical analysis of subgenomic mRNA (sgmRNA) processing revealed extensive variation in the number of sgmRNAs which may be generated during infection and in the lengths of noncoding sequence between leader-body junctions and the translation-initiating codon AUG. In addition, VR-2332 and LV select different leader-body junction sites from a pool of similar candidate sites to produce sgmRNA 7, encoding the viral nucleocapsid protein. The presence of substantial variations across the entire genome and in sgmRNA processing indicates that PRRSV has evolved independently on separate continents. The near-simultaneous global emergence of a new swine disease caused by divergently evolved viruses suggests that changes in swine husbandry and management may have contributed to the emergence of PRRS.     

Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid

The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (Aequorea victoria) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An analysis of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) analysis of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a generalized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was observed, confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.     

Isolation and stability of histidine-tagged proteins produced in plants via potyvirus gene vectors

A system for the expression and purification of histidine-tagged proteins from plants has been developed using a tobacco etch potyvirus (TEV)-derived gene vectors. The vectors offered a convenient polylinker and a choice of histidine tagging at the recombinant proteins' N or C termini. These vectors were utilized for expression of proteins encoded by beet yellows closterovirus (BYV). Approximately 4 micrograms/g of 20-kDa BYV protein was readily isolated from plants systemically infected by hybrid TEV. In contrast, only minute quantities of 22-kDa BYV capsid protein (CP) histidine-tagged at its N or C terminus could be purified. Rapid degradation of the recombinant CP has been implicated in its failure to accumulate in infected plants. Fusion with TEV HC-Pro stabilized the histidine-tagged BYV CP and facilitated purification of the fusion product from infected plants. This same fusion approach was successfully used with the 24-kDa minor BYV CP. The recombinant proteins were recognized by histidine-tag-specific monoclonal antibody in immunoblot analysis. These results demonstrate the utility of a designed series of TEV vectors for expression, detection, and purification of the recombinant proteins and suggest that intrinsic protein stability is a major factor in a recovery of recombinant proteins from plants.